乙型肝炎病毒表面抗原主蛋白基因转基因细胞系的建立与细胞性质的研究  

STUDIES ON A TRANSGENIC CELL LINE EXCRETING HIGH LEVEL OF HBsAg MAJOR PROTEIN

作　　者：任贵方[1] 阮薇琴[1] 田淑芳[1] 梅雅芳[1] 阮力[1] 杨安道[1] 杨芙蓉[1] 王申[1] 王秀平[1] 朱既明[1] 

机构地区：[1]中国预防医学科学院病毒学研究所

出　　处：《病毒学报》1991年第2期112-119,共8页Chinese Journal of Virology

摘　　要：使用本研究室以前构造的双拷贝乙肝病毒重组DNA质粒pSV_2DHBR2-32经改造构成pSV_2DHBR1-32。用此质粒转化CHO-dhfr^-细胞,经克隆、选择、加压增殖建立7个高产HBs-Ag的细胞系,其中首选B43对其生物学性状研究的结果说明,未发现微生物污染,无致瘤性,遗传稳定,纯度满意,核酸杂交试验说明该细胞是整合型,每个细胞约有200个左右的S基因拷贝,转瓶培养研究B43细胞分泌HBsAg的最佳条件为转瓶容积4升,表面积1300cm^2,细胞长成单层后换维持液150ml,其后每48小时收、换液1次,在36℃每小时8转,连续收液60天左右, 每升收液最高产HBsAg为7.5mg。此细胞可用作乙肝基因工程疫苗生产的候选细胞系。A new recombinant plasmid named pSV2 DHBR1-32 containing an Xho I -Bgl II fragment of S gene of hepatitis B virus was constructed by deletion of a 2.8 kb fragment of HBV DNA from the pSV2DHBR2-32 which is a pSV2 based plasmid containing two copies of the adr subtype of HB-sAg gene, previously reported by our laboratory. Seven CHO cell lines secreting the major protein of HBsAg at high level were developed by selection in increasing concentration of methotrexate after transformation with the new plasmid. Biological characteristics of one of the 7 cell lines, namely, the B43 cell line was studied. The results showed that B43 cell line was not contaminated by microorganisms including bacteria, fungi, mycoplasma and exogenous or endogenous viruses. It showed no tumero-genicity in nude mice. The genetic stability and purity of the cell population were confirmed. About 200 copies of S gene were detected in each cell in an integrated form.The kinetics and optimal conditions for HBsAg secretion from B43 cells were studied. Using 20 rolling bottles ( Forma ) in one cycle of production (2 months), changing the growth media to 5% calf serum instead of 10%calf serum after monolayer formation, culturing at 36℃ and 8 rph with 150ml of maintainance media per bottle, the yield was 7.5mg/L per 48hrs. Our conclusion is that the B43 may be a candidate vaccine production cell line.

关 键 词：乙肝病毒 主蛋白基因 转基因系 

分 类 号：R373.21[医药卫生—病原生物学]